N. V. Dovidchenko, E. I. Leonova, and O. V. Galzitskaya*

Received June 10, 2014; Revision received July 28, 2014
Amyloid and amyloid-like aggregates are elongated unbranched fibrils
consisting of β-structures of separate monomers positioned
perpendicular to the fibril axis and stacked strictly above each other.
In their physicochemical properties, amyloid fibrils are reminiscent of
synthetic polymers rather than usual proteins because they are stable
to the action of denaturing agents and proteases. Their mechanical
stability can be compared to a spider’s web, that in spite of its
ability to stretch, is stronger than steel. It is not surprising that a
large number of diseases are accompanied with amyloid fibril depositing
in different organs. Pathologies provoked by depositing of incorrectly
folded proteins include Alzheimer’s, Parkinson’s, and
Huntington’s diseases. In addition, this group of diseases
involves mucoviscidosis, some types of diabetes, and hereditary
cataracts. Each type of amyloidosis is characterized by aggregation of
a certain type of protein that is soluble in general, and thus leads to
specific distortions of functions of the corresponding organs.
Therefore, it is important to understand the process of transformation
of “native” proteins to amyloid fibrils to clarify how
these molecules acquire such strength and what key elements of this
process determine the pathway of erroneous protein folding. This review
presents our analysis of complied information on the mechanisms of
formation and biochemical properties of amyloid fibrils.
KEY WORDS: prion, stress granules, Alzheimer’s disease,
oligomer particles, thioflavin T, aggregation kinetics

The paradigm that one polypeptide chain can accept only one unique
three-dimension conformation became history long ago [1]. Convincing evidence of this are prions, which
illustrate that because of conformational changes, one protein can form
various structures including amyloids and amyloid-like fibrils. This
became especially relevant when it was clarified that incorrect packing
of some proteins could be a reason for pathological aggregations and
cause development of many neurodegenerative diseases such as
Alzheimer’s and Parkinson’s, type II diabetes, amyotrophic
lateral sclerosis (ALS), frontotemporal degeneration (FTD),
Huntington’s disease, etc. [2-5]. It should be noted that not all amyloids and
different amyloid-like fibrils are associated with neurodegenerative
diseases, and this property is inherent in a great number of proteins.
However, in spite of such diversity of proteins, at first glance the
formed amyloid fibrils are similar and look like antiparallel
β-structures stacked in a pile perpendicular to the fibril axis
[6]. Electron microscopy and X-ray analysis have
revealed a more accurate description of fibrils: amyloids are
antiparallel β-sheets folded in a helix with a cylinder-like
cavity formed within the latter [7]. Amyloid-like
structures are formed by yeast prions Sup35 that are rich in
asparagines and glutamines. Nevertheless, more profound analyses have
revealed a number of differences between amyloid fibrils depending on
the protein composition and conditions under which they are formed. For
example, α-synuclein, aggregates of which are specific for
Parkinson’s disease, forms single cylinders consisting of
β-sheets, while fibrils of Aβ-fragments specific for
Alzheimer’s disease consist of two- or three-cylinder
β-sheets [7].

The first amyloid depositions were detected in tissues upon iodine
staining more than 150 years ago [8]. The
depositions were stained blue analogous to starch grains in plants.
Though the result lead to the erroneous conclusion of the existence in
humans of starch grains like in plants (hence the name
“amyloid” appeared), during the following 100 years data
were accumulating on the occurrence of amyloids in different tissues.
At the time of the discovery of amyloid depositions, researchers could
use only a light microscope and various stains, and during the
following “descriptive” period the revealed depositions
were classified depending on the site of location, the extent of
staining with a certain reagent, and the clinical case in which the
amyloid depositions were found. Therefore, in the 1920s it was known
that amyloids were stained with Congo red and therefore they could be
attributed to active substances, and the extent of staining indicated
their amount in the sample [9, 10]. For example, Congo red was once used as a test
for amyloidosis, when the stain was applied intravenously, and after a
specified time the portion of bound stain in serum was estimated [11, 12]. It was accepted that
the estimate was proportional to the total amount of amyloids in the
blood. Though the use of the test was discontinued because of the risk
of developing anaphylactic shock, it was believed that the binding of
amyloid aggregates is a specific peculiarity of the stain, and it was
used for isolation of fibrils from depositions upon autopsy. However,
at present the accurate mechanism of binding with Congo red remains
unclear [13, 14]. Another
frequently used stain for revealing amyloidosis is thioflavin T (ThT),
which when incorporated in fibrils significantly increases the
fluorescence quantum yield with a shift to longer wavelength of the
spectrum. However, not all amyloid formations are stained with these
preparations, including protein MAVS that is active in antiviral
immunity [15], α-synuclein in
Parkinson’s disease [16], τ-protein in
Alzheimer’s disease [17], FUS in amyotrophic
lateral sclerosis [18], and others. This can be
explained by the fact that in water the short-wavelength excitation
spectrum overlaps insignificantly the absorption spectrum of the stain,
i.e. only weak absorption occurs on maximal excitation. Meanwhile, for
a long-wavelength excitation spectrum, thioflavin T fluoresces in the
same spectral range as the stain incorporated in fibrils, the solution
of the stain and amyloid fibrils being usually a mixture of unbound
stain and stain intercalated in the fibrils [19].
It was demonstrated that thioflavin T can bind to aromatic amino acid
residues located in a particular order in β-sheets [20]. Although thioflavin T is widely used as a
fluorescence probe, no agreement has been achieved on the mechanism of
thioflavin T incorporation into amyloid fibrils [19].

FORMATION OF AMYLOIDS BY THE PRION MECHANISM

Prions are proteins able to accept different conformations including
amyloid fibrils that can serve as a matrix and “infect”
other proteins, both within and between cells and between organisms [21-23]. Originally, prions were
studied in connection with infectious diseases such as Kuru,
Creutzfeldt–Jakob disease (CJD), scrapie, bovine spongiform
encephalopathy, fatal familial insomnia, in which a prion protein was
the pathological agent [24]. However, later it was
found that yeast prions assist them in adaptation to diverse conditions
of the environment [25]. In mammals they can both
cause different diseases, sometimes even leading to death, as well as
performing useful functions, for example, activate cell-based innate
immunity (protein MAVS), provide for long-term memory (protein CPEB)
[26, 27], and form stress
granule assemblies under conditions unfavorable for the cell [28]. Many proteins contain prion-like domains that
endow the protein with properties close to those of prions, including
the ability for self-assembly. For example, the amino acid composition
of prion-like domains of proteins fused in sarcoma (FUS), TAR
DNA-binding protein-43 (TDP-43), and cytotoxic granule-associated
RBA-binding protein (TIA1) is similar to prion domains of yeast
proteins such as Sup35 and Ure2 [29]. The latter
two proteins are characterized by a high content of polar amino acid
residues, for example, asparagine, glutamine, tyrosine, and glycine.
These domains enable the proteins to pass from the unfolded
three-dimensional structure to intermediate states, which in their turn
are predisposed to different conformational transitions including the
formation of amyloid fibrils [18, 30, 31]. These proteins are
generally in a dynamic equilibrium between two forms: unfolded soluble
monomers and oligomers close to molten ones. Such oligomers can be
involved in diverse conformational states (Fig. 1).
According to one scenario, they can organize in structured
amyloidogenic oligomers, then transform into pathological aggregates of
a non-amyloid type or amyloid fibrils. The latter can serve as a
“matrix” for incorrect folding by the prion type. According
to another scenario, protein molecules can form amorphous aggregates
consisting of both soluble monomers and molten oligomers organized as
dynamic cross-β-structures with properties of fluids, and can also
form gel-like structures [32-34]. The hydrogel state is crucial for the formation
of various non-membrane structures such as stress granules, ribonucleic
complexes, etc. [31, 32, 35, 36]. In other words,
prion-like domains are required for aggregation at the stage of protein
conformational transition from the fluid state to the gel-like state in
order to perform some vital functions, but under certain conditions
they can irreversibly transform into amyloid fibrils.

Fig. 1. Schematic representation of prions and prion-like domains
that can lead to different conformational states and scenarios
(modified and adapted from [37]).

FORMATION OF AMYLOIDS BY THE PRION MECHANISM RESULTING IN
PATHOGENESIS

Recently, a new concept on the development of some neurodegenerative
diseases has appeared. It includes the idea of the transition and
multiplication of protein aggregates from cell-to-cell, from one cortex
lobe to another during the disease progression. It has been shown both
in vitro and in vivo that in different neurological
diseases, pathological proteins can invade into a cell, find proteins
similar to themselves, and then serve as a “matrix” for
pathological aggregation [23, 38-41]. Thus, pathologic
affection of cells spreads from the lesion focus. It should be noted
that the cases when the disease broke out in different cortex lobes do
not necessarily exclude the prion-like mechanism of its excrescence. In
such a case, every lesion focus would spread the disease independently
from the others. But what is the molecular basis for neurodegeneration
spreading by the prion mechanism? How can these proteins acquire the
conformation when the modified protein can serve as a
“matrix” for “healthy” proteins, transforming
the latter into an aggregated state?

The formation of insoluble β-amyloid fibrils in brain tissues
accompanies many neurodegenerative diseases such as Alzheimer’s
disease, Down’s syndrome, and others. The Aβ-peptide is
formed as a result of proteolytic cleavage of the amyloid precursor
protein (APP). The mRNA of APP frequently undergoes alternative
splicing, and so it has several isoforms. In general, proteins perform
important physiological functions, and the APP gene is expressed in
almost all cells. A change in APP proteolysis results in accumulation
of Aβ-fragments that associate in amyloid fibrils [42].

When prion-like domains were discovered in TDP-43 and FUS [18, 30, 31,
43], it became clear that mutations in these
proteins can lead to pathological states. Thus in protein TDP-43,
mutations Q331K and M337V located in the prion-like domain directly
enhance incorrect folding. In protein FUS, mutations associated with
amyotrophic lateral sclerosis (ALS) are positioned in the nuclear
localization signal (NLS), which causes the protein to accumulate in
the cytoplasm, thus facilitating pathological aggregation. Normally,
the FUS protein is localized in the nucleus, though it sometimes moves
to the cytoplasm and back [44-49]. Along with TDP-43 and FUS, prion-like domains
have been revealed in 40 other RNA-binding proteins. It is not
surprising that they will be also related to the development of ALS and
similar neurodegenerative diseases. As has been determined, other
members of the family such as TAF15 (TATA box-binding protein
(TBP)-associated factor) and EWS (Ewing sarcoma breakpoint region 1)
are also connected with ALS and FTD. Moreover, in the case of FTD
another RNA-binding protein, PSF (PTB-associated splicing factor), is
anomalously accumulated in the cytoplasm of oligodendrocytes and forms
an insoluble structure [50]. Mutations in TIA1,
which is an important protein for the formation of stress granules,
have been detected in Welander distal myopathy (a slowly progressing
muscle dystrophy) [51]. In RNA-binding proteins
with prion-like domains, other mutations associated with different
neurodegenerative diseases were also revealed [52]. This suggests the important role of this domain
in pathogenesis.

FORMATION OF AMYLOIDS BY THE PRION MECHANISM IS REQUIRED FOR
NORMAL CELL FUNCTIONING

Living organisms widely use the property of protein molecules to form
amyloid structures for various specific purposes. Normally, some
organisms form amyloid fibrils for performing various functions. One of
the best-studied examples of such functional amyloids is protein
curlin, which is used by E. coli to colonize inert surfaces and
is a mediator upon binding with proteins of other organisms. Like other
amyloid structures, such fibrils are 6-12 nm thick in diameter,
have a large portion of β-structure (as shown with CD), and bind
thioflavin T and Congo red [53]. Another example
is the bacterium Streptomyces coelicolor, which due to the
formation of amyloid fibrils with chaplin proteins form hyphae used for
spreading spores [54].

In these examples, the process of amyloid nucleation that will initiate
aggregate growth depends on the ambient conditions and is controlled by
a definite cascade of reactions. Controlled formation of functional
amyloid aggregates occurs in mammals as well. For example, melanosomes
are organelles differentiating into melanocytes responsible for melanin
biogenesis in skin cells, containing fibril formations on which melanin
granules are formed. Such fibril formations have much in common with
amyloids; they are formed from a proteolytically cleaved domain of the
membrane protein Pmel17 [55].

Long-term memory is also provided by the principle of fibril formation
in which protein CPEB (an RNA-binding protein capable of controlling
local translation of mRNAs in dendrites) plays an essential role. This
protein can stimulate mRNA polyadenylation, and its aggregation
activates translation of the “silent” mRNA accumulated in
synaptic end-feet [56]. The N-domain of CPEB is
rich in asparagines and glutamines, which is specific for prion-like
domains.

Thus, protein MAVS located on the surface of mitochondrial membranes
activates innate antiviral cell immunity. When aggregated, it can
interact with cytoplasmic receptors recognizing patterns specific for
most pathogens, which activates a cascade of reactions leading to the
synthesis of β-interferon [57, 58]. This protein can also aggregate by the prion
mechanism.

These examples demonstrate that even in highly organized organisms, the
formation of amyloids located in a strictly defined place and rigidly
controlled can be beneficial physiologically for performing specific
and specialized biological functions.

BIOCHEMICAL CHARACTERISTICS OF AMYLOID FIBRILS

In the first stages of studies, chemical analysis of amyloids was
complicated because both in human and animal samples the formed fibrils
are frequently coated with other tissue components resulting in their
nonsusceptibility to the extraction procedure. More vigorous methods of
extraction, such as the use of strong bases and acids, destroy amyloid
structures. The crucial point in chemical identification of amyloid
proteins was the fact that intact amyloid fibrils can be quantitatively
extracted from human and animal tissues using a distillate and buffers
with low ionic strength [10]. The procedure is as
follows: the tissue samples are repeatedly extracted with a saline
solution. When the supernatant stops showing significant optical
density at 280 nm, the granules are kept in the distillate until
the formation of an opalescent solution that upon precipitation with
salt leaves a precipitate of pure fibrils associated with Congo red
when viewed in an electron microscope. Then the fibrils can be
denatured in 8 M urea or 6 M guanidine chloride and layered
onto a chromatography column for further purification to analyze the
amino acid sequence [59, 60].
It is evident that any product soluble in the saline solution will be
lost upon washing, and so will impede the determination of critically
important molecules that lead to amyloidogenesis or toxicity. The
chemical identification of specific precursors allowed the development
of immunohistochemical reagents that can identify the chemical nature
of amyloids in tissue samples without chemical extraction.

The first identified and chemically characterized amyloid was an
aggregate of light chains of human immunoglobulin isolated from tissues
of a patient with so-called primary amyloidosis and was not associated
with any other disease [61]. Subsequent studies
showed that the same class of proteins is a precursor of amyloid
depositions in multiple myeloma [62]. L-chains and
their fragments included in amyloid have a similar amino acid sequence
with circulating in serum and/or monoclonal light chains isolated from
the same patient. The depositions in tissues consist of intact L-chains
and/or C-terminal fragments of such chains, which shows that the
fragments in vivo subjected to proteolysis are more
amyloidogenic than intact chains or the intact chain depositions are
the object of proteolysis in situ [63].
Biosynthetic experiments with bone marrow cells revealed that in some
cases truncated L-chains can be products of the synthesis, but these
experiments were not strict enough to exclude the hypothesis of a fast
postsynthetic digestion [64]. Subsequent
experiments demonstrated that fragments of some isolated L-chains
obtained by treatment with proteases in vitro formed
amyloid-like fibrils in a test tube [59, 65]. The basic result of these experiments is the
finding that the amyloidogenic potential is associated with the amino
acid sequence of the protein.

The following observations revealed some structural peculiarities
rendering some members of a given class of proteins more amyloidogenic
as compared to other members [66]. Experiments
with recombinant versions of the variable region of the amyloidogenic
IgL-chain and non-amyloidogenic IgL-chain (obtained from an
amyloidogenic molecule by mutating some amino acids) also showed
differences that can lead to the propensity to form fibrils [67]. From the biophysical point of view, the
multiplicity of sequences of amino acid residues in L-chains brings
about four potentially soluble forms when the concentration of any
single L-chain molecule increases by cloning upon the immune response
during the disease.

Some human L-chains of immunoglobulins (about 15% of all possible
versions of L-chains, predominantly from the λ-class) are
exceptionally amyloidogenic and form deposits leading to dysfunction of
many organs, especially kidney, heart, stomach, and peripheral nerves
[63].

Other L-chains can form non-fibrillar aggregates, and this is a menace
for organs just like the fibril-forming L-chains [63]. Cases of depositions of the two types of the
same protein in one person are rare, though having been registered, and
this suggests that the conformation acquired in vivo can be
associated with the environment of the affected tissue [68].

Still other L-chains have a specific Bence-Jones feature (aggregation at
specific temperatures, usually 56°C, with subsequent dissociation
at higher temperatures), and this is associated with aggregation in
renal tubules where the protein concentration is higher while pH is
lower, and this can lead to kidney dysfunction [69].

Another fraction of L-chains of human immunoglobulin probably does not
precipitate in physiological conditions and does not aggregate in
vivo [70]. The exact structural features
responsible for the wide multiplicity of biophysical peculiarities of
proteins so similar under physiological conditions are not known.

It is no surprise that taking into account the homology of Ig domains,
rare cases have been reported when solitary heavy chains served as
precursors for amyloid aggregates upon monoclonal B-cell production [71]. The initial non-immunoglobulin amyloid was
isolated from depositions in a monkey with chronic inflammation. The
protein has an amino acid sequence dissimilar either to the sequence of
an amyloid formed by immunoglobulin L-chains or to the sequence of any
other protein sequenced to date [72]. At the same
time, a similar protein was isolated and sequenced from the kidney of
an Israeli citizen with a periodic disease and secondary (or associated
with inflammation) amyloidosis [60]. The two
proteins had similar amino acid sequence, it being identical from the
N-terminus and varying amino acids not deviating from the frequency
characteristic of residues of normal polymorphisms. Later it was found
that the given protein also forms amyloids under long-term rheumatoid
arthritis (another chronic inflammatory disease) and is a basic
component of fibrils in murine and rat models [73].

More than 30 amyloid proteins are now recognized by the International
Nomenclature Committee on Amyloidosis [33, 50, 74, 75]. The precursors of most of these proteins are
apolipoproteins A (1, 2), serum amyloid A, as well as different
proteohormones and immunoglobulins [76]. It has
been found that depositions contain also other molecules such as
apolipoprotein E (ApoE), SAP (serum amyloid P component), and the
proteoglycan perlecan [77-80]. Under AA-amyloidosis (see below), fibrils are
generated from the cleaved product SAA whose concentration increases
upon development of chronic inflammations and other systemic diseases
[81, 82]. This protein is
involved in lipometabolism in macrophages participating in
inflammations and can also realize other functions, for example, play a
part in atherogenesis associated with inflammation [74]. SAA was the first of the five proteins (the
other proteins being IAPP, Aβ-peptide, prion, and protein Bri2
whose depositions were found at fatal familial insomnia [83-86]) in which amyloid
depositions provide evidence of a normally functioning soluble
precursor, identified either after preparation of appropriate
antibodies to fibrils and the binding of these antibodies to normal
protein, or after identification using cDNA of the corresponding gene
in normal tissues.

However, of more importance is that these investigations allowed
developing isolation methods that can be used for studying fibrils from
any tissue or organism; for example, the methods were used to isolate
proteins from congophilic vascular and cerebral deposits of Aβ [87, 88]. Extracellular molecules
of matrix and membrane components like laminin and tenascin were also
present in some depositions. Such molecules may contribute to
accumulation and formation of depositions in tissues. It has been
proposed that additional molecules can play a part in stabilization of
fibril structure or provide conditions for enhancement of
fibrogenesis.

EXPERIMENTAL MODELING OF AMYLOIDOSIS IN ANIMALS

Since the 1960s, animal models have become one of the means to study
amyloidosis. By introducing a particular dose of casein or
Freund’s adjuvant into mice or rabbits, an acute inflammatory
process was provoked which, in turn, was concomitant with accumulation
of deposits stained with Congo red in the liver, kidneys, and spleen
[89]. It was found that as a result of
inflammation, the generation of cytokines promotes formation of the
amyloid precursor serum amyloid A (SAA) [90]. This
model completely mirrors human AA amyloidosis generated during
inflammations and is frequently used for studying pathogenesis. A high
percentage of amyloidosis in humans is associated with AA amyloidosis,
so-called secondary or reactive amyloidosis, when as a response to any
chronic inflammation, acute phase proteins (precursors of serum amyloid
A – SAA) are synthesized in the liver [76]. Another interesting feature of the model is that
spleen extracts of an already infected animal, when transferred to
another animal, can competitively accelerate the process of fibril
deposition. Such acceleration was explained by the presence of fibril
fragments that can serve as beginnings for genesis and deposition of
newly formed amyloid precursors [91]. By
associating monomers to the termini, these seeds can form elongated
protofibrils, which later can form amyloid fibrils. Of interest is that
the murine model of amyloidogenesis can be activated by incorporating
seeds of xenogenous origin, which shows that the seeds have comparable
structural elements [92, 93].
A similar phenomenon was found for the model of spontaneous
amyloidogenesis in SAMP1 mice. This line is characterized by the
existence of an isoform of protein ApoA2(C) with the following
substitutions: Gln for Pro in position 5, Ala for Val in position 26,
and Met for Val in position 38 [94]. The rate of
amyloid accumulation in the animals is directly proportional to their
age and, as has been revealed, amyloid accumulation in young animals
can be accelerated by introduction of preparations obtained from aged
animals [95, 96]. These
models are characterized by the requirement of a sufficient amount of
the amyloid precursor, caused genetically in one case (ApoA2-AA
amyloidosis) and by artificially stimulated inflammation in the other
case of AA amyloidosis. Mutual effect of ApoA2-AA is also likely upon
either inhibition or acceleration of fibril formation [97].

Protein ApoE is no less important in the formation of amyloid fibrils.
The progeny produced by cross breeding of
apoe-(ApoE–/–) knockout animals with transgenic mice
with hyperexpression of mutant human protein APP-(PDAPP+/+) contained
much less cerebral Aβ deposits compared to the transgenic parents
(PDAPP+/+ ApoE+/+) [98, 99].
As a result of these experiments, it was suggested that protein ApoE
might be used for the treatment of Alzheimer’s disease [100]. However, further experiments with apoe
knockout mice demonstrated that such animals are sensitive to
amyloidosis caused by IAPP to the same extent as the wild-type mice.
There are conflicting data on the increase or decrease in depositions
in inflammation-induced AA amyloidoses [101-103]. It was shown that protein SAP in vitro
inhibits cleavage of amyloid fibrils not because of direct inhibition
of the process, but rather because of direct binding to fibrils, thus
shielding potential cleavage sites from proteases [80]. The sap knockout mice had a lower rate of
amyloidogenesis in response to inflammatory stimulation, which agrees
with the above observations, but these experiments do not indicate that
protein SAP is required for fibrogenesis [104, 105]. The absence of sustainable models with
knockout of the gene encoding perlecan prevents experiments like those
with SAP protein. At the same time, the observations of expression of
the gene encoding perlecan before the production of amyloid depositions
in a murine model of AA-amyloidosis in tissues, as well as the
experiments in vitro, demonstrated the acceleration of
fibrillogenesis by the action of both the core protein perlecan (a
heparan-sulfate proteoglycan) and associated with it chains of heparan
sulfate and chondroitin sulfate. In particular, the formation of
amyloids in transgenic mice with hyperexpression of heparanase was
inhibited. Moreover, other sulfated glucosoaminoglycans, such as
heparin, dermatan sulfate, dextran sulfate, and pentosan, augmented the
formation of amyloid fibrils [106-109].

The use of these models shows that homomolecular aggregation associated
with nucleation and the subsequent growth of fibrils occurs in
connection with heteromolecular interactions, some of which are
amyloidogenic and others are anti-amyloidogenic.

Monitoring of these models permitted posing the important question
whether all amyloids are prion-like (or possess infectious activity)
and whether this phenomenon can hold true for some forms of amyloids
causing neurodegenerative diseases capable of transmission over the
whole organism [110]. However, one should take
into account the difference between cell-to-cell transmission of
amyloids in a restricted space where autologous proteins and genetic
compatibility for direct cell-to-cell transmission exist and the spread
of infectious diseases from the environment, which imposes strict
requirements on the amount and quantity of interactions between the
seed and further monomers of the aggregate. The difference between
prions existing predominantly as fibrils and infectious prions as well
as the visible interleaving of the aggregation and neurotoxicity phases
during crisis may reflect in some way acquisition or selection of
conformational compatibility required for infectivity or toxicity [111, 112].

CHARACTERIZATION OF KINETICS OF AMYLOIDOGENESIS

To make the description of amyloidogenesis complete, it is necessary to
find all possible conformational states of the protein during its
aggregation as well as possible oligomeric structures formed during
amyloidogenesis. Moreover, the description would not be complete
without a corresponding determination of kinetic and thermodynamic
parameters for all potential forms and states of the protein in the
course of aggregation. It should be noted that these requirements
include also the analysis of aggregation on the molecular level for
subsequent revealing of the regions vital for the whole process of
amyloidogenesis. It is accepted that this process has a nucleation
stage (see Fig. 2). The aggregation of protein
monomers into a fibril can be divided in two stages: the lag-time when
seeds are generated, and the time of transition of all monomers into an
aggregate. Notice that the latter phase varies depending on the protein
and can be either linear or exponential [113].
Reactions having a nucleation stage have been studied quite well from
the theoretical and experimental points of view for a wide range of
substances, mostly in connection with crystallization of large and
small molecules [114].

Fig. 2. Schematic representation of amyloidogenesis.
km+ is the rate constant of monomer association with
the oligomer; km– is the rate constant of
monomer dissociation from the oligomer; kn+ is the
rate constant of transition of the seed (an oligomer consisting of s
monomers) from state α to state β; kp
is the polymerization rate constant; kexp is the rate
constant of exponential increase of the ends of a growing
fibril.

Like for other processes with a nucleation phase (including
crystallization), the addition of primes from the seeds already formed
during amyloidogenesis practically levels off the lag-period because
the aggregation rate is no longer limited by the nucleation stage [115, 116]. It was demonstrated
that introduction of particular mutations to aggregating proteins or
certain changes in experimental conditions could also level off the
lag-period, assuming that nucleation is not a limiting stage of the
process any longer [117-119]. The absence of a lag-period is not necessarily
related to the fact that the description of the aggregation mechanism
with a nucleation stage is no longer applicable. Rather, it shows that
the time required for fibril formation is quite large relative to the
rate of seed formation, and so nucleation is not already a
rate-limiting stage during the transition of the protein from the
soluble form to an amyloid. Though no fibrils appear in the lag-period,
it is clear that this is an essential stage for the formation of
different oligomers including those that would serve as a seed for the
formation of mature fibrils.

The efficiency of the fibrils formed in stimulation of aggregation as a
seed can strongly decrease on an increase in the differences of the
primary structure [120-122]. It was demonstrated for the example of
immunoglobulin domains with different primary structure that
co-aggregation of various types of domains does not occur when the
identity of the protein primary structure is below 30-40% [121]. Bioinformatics analysis of homologous
sequential domains in large multidomain proteins revealed identity of
less than 40%, which may be evidence of the primary structure of such
proteins being composed to avoid aggregation.

During recent decades, significant attempts have been made to identify,
isolate, and describe oligomeric particles formed in solution before
fibrils appear. This interest in oligomers can be explained by two
reasons: apparently, the formation of such particles is a vital stage
in amyloidogenesis, and one has every reason to believe that oligomers
are the greatest menace for pathogenesis, i.e. they are toxic. As an
example, we can take the formation of amyloids by the Aβ-peptide.
The aggregation of this peptide is preceded by the development of a
number of metastable non-fibril formations observed using the AFM and
TEM techniques [123-126].
Some of the formations look like small spherical beads 2-5-nm in
diameter, and others look like small beads on a string with individual
beads having also the diameter of 2-5 nm, and the third form ring
structures that have evidently appeared as a result of closure of the
structures similar to the beads on a string. All these assemblies,
called protofibrils by the authors who were the first to detect them
[123-126] should not be
confused however with protofilaments that are singular threads of
mature fibrils. Protofibrils, which consist of the Aβ peptide, can
bind Congo red and thioflavin T [126], contain a
large portion of β-structure and are composed of about 20
molecules forming small spherical particles.

Similar spherical and bead-like protofibril formations were also
detected in other systems including α-synuclein [127], amylin [128],
immunoglobulin light chains [129], transthyretin
[130], poly-Q [128],
β2-microglobulin [131], lysozyme
[132], acylphosphatase from Sulfolobus
solfataricus [133], and the SH3 domain [134]. These formations can be characterized as
particles rich in β-structure with sufficiently high orderliness
allowing for binding Congo red and thioflavin T. A peculiarity of the
formations is the affinity of specific antibodies to protofibrils
obtained from different sources, the absence of affinity to monomer
units and mature particles, which suggests the presence of definite
similar structural elements in soluble oligomer formations.

In some cases protofibrils may be off pathway aggregates [131, 135], but there are data
showing that protofibrils represent a state that the protein overcomes
during amyloidogenesis [116, 123]. It was found that the protofibril →
mature fibril transition of peptide (109-122) from Syrian hamster prion
protein proceeds through the adjustment of originally non-aligned
β-regions forming a potential fibril [136].
This alignment includes isolation of β-regions with their
subsequent inclusion into the potential fibril; however, internal
rearrangement of β-regions is also possible, which is realized
subject to conditions [137]. Summarizing the
above, it can be stated that independent of the particular role of
protofibrils in the formation of amyloid fibrils, the determination of
the mechanism of formation of such oligomer particles as well as their
structure is extremely important especially because it is believed that
amyloid fibrils are the basic toxic formations involved in
neurodegenerative diseases.

Further investigations of oligomer particles preceding the formation of
fibrils have become feasible through the method of photoinduced
coupling of unmodified proteins (PICUP). Thus, it has been found that
soluble oligomers formed by the Aβ-peptide (both versions 1-40 and
1-42) exist in dynamic equilibrium with the monomer form. These
oligomers consist of 2-4 monomers for Aβ 1-40 and 5-6 monomers for
Aβ 1-42 and, as shown by CD methods, are relatively unstructured
[138]. The interest in oligomer formations of
Aβ was driven, in particular, by detection of such particles in
the brains of patients with Alzheimer’s disease [139] and also in lysates and the solution
concentrate with cells expressing the precursor protein of Aβ [140, 141].

Region NM of yeast prion Sup35p is liable also to fast formation of
unstructured oligomers. Transition with a growing β-structure
takes place only after the formation of such oligomers, and then after
transformation they can serve as seeding for fibrils [116]. The transition can occur due to covalent
dimerization of NM molecules if the residues in the head of particle N
(25-38) are cross-linked. Moreover, provided the oligomers are kept in
an oxygen-enriched medium, intramolecular disulfide bridges form more
promptly for the molecules in which cysteines are in the N region.
These results apparently show that the interaction of the two head
regions of molecule N generates a seed for an amyloid-like structure
within the aggregate.

Similar behavior was detected for aggregation of denatured yeast
phosphoglycerate kinase at low pH using dynamic light scattering and CD
spectroscopy [142].

In this case, stabilization of β-structure occurred with growing
dimensions of the aggregate. When a critical mass is accumulated,
oligomers stick together and form short irregular protofibrils similar
to those formed by Aβ and α-synuclein [142]. Moreover, the unfolding of the SH3 domain of
bovine phosphatidylinositol kinase leads to fast formation of
unstructured particles of different dimensions, which then transform
sequentially into thin irregular protofibrils that bind thioflavin T
[134]. So, the experimental data show that
structured protofibrils can form either by sticking to each other or
through rearrangement of small relatively unstructured oligomers formed
at the very beginning of aggregation.

CONCLUSION

Many natural proteins can form amyloid fibrils. Nevertheless, the
molecular mechanism underlying the formation of amyloids is still
unclear. This is because a vast number of factors can affect the
conformational transition from a “native” protein into
pathological aggregates, including high protein concentration, specific
proteolytic cleavage, mutations, interaction with ligands, and many
other factors not yet determined. At present, many questions concerning
the process of protein aggregation remain unanswered. A striking
circumstance is that amyloidogenesis is specific for all living beings
from microorganisms to humans. Based on this, it can be concluded that
formation of amyloid fibrils has been tested by evolution and has
preserved a conservative structure. The unique physicochemical
properties of amyloids and the fact that many proteins form them with
an ability to regulate the process as well as its being widespread in
nature highlight the biological requirement for such formations. In
spite of a great variety of neurodegenerative and other diseases
associated with the formation of pathological protein aggregations, it
can be proposed that the ability of proteins to form amyloid aggregates
is connected first of all with multiple functions of proteins, and
therefore the library of proteins generating “functional”
amyloids will widen. Further studies are required to clarify the
detailed mechanism of fibril amyloidogenesis and to reveal certain
determinants affecting the kinetics of the process. But first and
foremost is determination of possible key factors for controlling the
process of aggregation to prevent development of pathologies and
diseases caused by them, or on the contrary to use them for
amelioration of health, for example, for improving antiviral immunity
or memory strength.

This study was supported by the Russian Science Foundation No.
14-14-00536.